Gamma-Secretase is an unusual enzyme that catalyzes the intramembrane proteolysis of numerous type I integral membrane proteins. These include the amyloid-beta protein precursor (APP), which releases the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD). For this reason, gamma-secretase is a prime target for the development of therapeutic and preventative agents for AD. Pharmacological, genetic, and biochemical evidence generated under this project strongly supports the hypothesis that the polytopic membrane protein, presenilin (PS), is the catalytic component of a larger multi-protein gamma-secretase complex. PS is normally cleaved into two fragments that remain associated. These heterodimers are metabolically stable, and their formation is tightly regulated by limiting cellular factors. During the current project period, we discovered that both PS endoproteolysis and gamma-secretase activity require two conserved transmembrane aspartates, consistent with our provocative hypothesis that presenilin undergoes autoproteolysis to become the catalytic component of a novel aspartyl protease. Moreover, using affinity isolation on a designed inhibitor, we showed that the PS-associated protein, nicastrin, is another member of the complex. Recently, our laboratory has found that the co-expression of PS1, nicastrin, and two proteins identified in C. elegans, aph-1 and pen-2, results in increased PS heterodimer formation, full nicastrin maturation and enhanced gamma-secretase activity. All 4 proteins co-immunoprecipitate and bind to a gamma-secretase affinity matrix. Taken together, these new finding suggest that PS, nicastrin, aph-1, and pen-2 assemble as a complex that leads to the formation of active gamma-secretase. In light of these discoveries, we now propose to purify, fully characterize and then reconstitute active gamma-secretase. Specifically, we will: 1) scale up and refine the multi-step purification protocols for gamma-secretase established by our labs in order to unequivocally confirm the presence of these 4 key components and search for any additional protein factors by mass spectrometry; 2) systematically explore phospholipid, detergent, ionic, energy, pH and other requirements to optimize our established in vitro cleavage assay (which can generate Abeta, AICD and NICD) and characterize the kinetic properties of the protease; and 3) using this new knowledge, carry out the step-wise addition of recombinantly expressed PS, nicastrin, aph-1, and pen-2 in a pure detergent/phospholipid environment to reconstitute proteolysis. The purification, detailed characterization, and reconstitution of gamma-secretase will aid the understanding of this essential protease in health and disease and the search for effective and safe AD therapeutics. Moreover, this work will advance our fundamental knowledge of a unique new class of intramembrane-cleaving proteases.